Is your protein sticking to the membrane or crashing/aggregating from
the concentrating?
Try to find a buffer condition in which your protein is more soluble
and less aggregation prone. You can try varying salt concentration,
pH, and inclusion of additives like glycerol or detergent.
Ho
UC Berke
bulletin board on behalf of Sophia Tsai
> Sent: Wed 5/6/2009 7:12 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Lost my protein
>
> Hi everyone,
> It seems that our lab has experienced issues with precipitation of some
> proteins (not all) with the Amicon concentrator
@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Lost my protein
Hi everyone,
It seems that our lab has experienced issues with precipitation of some
proteins (not all) with the Amicon concentrators. For those, we have started
using VivaSpin concentrators. That seems to work fairly well with those that
have issues with
Dear Zhang,
Perhaps you can try to concentrate your protein using PEG2.
Put your protein into dialysis tubing and bury it with PEG2. You should
check it every 30-60 min.
Sincerely,
Li
Hi all,
during concentrating my protein, using Amicon Ultra centrifugal filter
Hi everyone,
It seems that our lab has experienced issues with precipitation of some
proteins (not all) with the Amicon concentrators. For those, we have started
using VivaSpin concentrators. That seems to work fairly well with those that
have issues with the filters. You may want to give it a try.
PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Lost my protein
Hi.
I have experienced similar problems with the Amicon filters previously. I
found that using a swing bucket rather than a fixed angle centrifuge and
keeping the speed at 4000 g, the recommended speed, resulted in better
yields
Hi.
I have experienced similar problems with the Amicon filters previously. I
found that using a swing bucket rather than a fixed angle centrifuge and
keeping the speed at 4000 g, the recommended speed, resulted in better
yields. If you have a large volume to concentrate (>12 mL), using an
Amico
Hi Yanming,
I usually keep the follow through before I know nothing is in it. Also
you may want to suspend your protein solution every 5-10 min to avoid
precipitation because the concentration gradient forms rapidly during
centrifugation. If all of these do not help, just try concentrators
wit
Hi Yanming,
you can try adding 1 M of urea, 1% detergent (OG,DM) that often helps to keep
proteins in solution.
Best wishes
Kornelius
On Tue, 5 May 2009 09:06:09 -0700
yanming Zhang wrote:
> Hi all,
>
> during concentrating my protein, using Amicon Ultra centrifugal filter
> devices, 5000g
, 2009 11:06 AM
Subject: [ccp4bb] Lost my protein
Hi all,
during concentrating my protein, using Amicon Ultra centrifugal filter
devices, 5000g, 4C, I lost large amount of my protein (75%). I heard the same
story from one of my colleagues too. It seems the membrane of the Amicon
Hi,
assuming that 1) you used the right MW cut-off and 2) the membrane is
intact, you probably "pelleted" your protein onto the membrane.
This often happens if the protein has a limited solubility in the buffer
you are using.
Remember, while concentrating your protein, you are actually "trave
Hi all,
during concentrating my protein, using Amicon Ultra centrifugal filter devices,
5000g, 4C, I lost large amount of my protein (75%). I heard the same story from
one of my colleagues too. It seems the membrane of the Amicon tube ate my
protein.
Why this happen and how to recover my prote
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